This study explores the role of the high affinity zinc site in the NR2A subunit of N-methyl-D-aspartate (NMDA) receptors in glutamate-induced neuronal cell death (i.e. excitotoxicity). Excitotoxicity is implicated in a variety of neuropathological conditions, including stroke, seizures and Alzheimer's disease. Effective treatment for some of these diseases is still lacking. A long-term objective of this study is to identify novel targets for developing new therapy. This laboratory has shown that an allosteric interaction between the high affinity zinc site in the amino-terminal domain (ATD) and the glutamate-binding domain of NR2A causes fast zinc-dependent desensitization. The role of zinc-dependent desensitization will now be studied with a "knock-in" mouse model in which the zinc site is abolished by a point mutation (NR2AH128A). The mechanism and structural determinants of this allosteric interaction will be studied in an artificial expression system (HEK293 cells). The receptor function will be assessed by whole cell patch-clamp recording and single channel recording. To probe the structural determinants, the ATD and the linker regions to the transmembrane domains will be mutated systematically. The following specific aims will be addressed: (1) To test the hypothesis that the allosteric interaction between the zinc binding site and the glutamate binding site reduces excitotoxicity without significant alteration of normal synaptic NMDA responses;(2) To test the hypothesis that the ATD of NR2 strongly influences the subtype-specific gating of NMDA receptors;(3) To identify the critical structural element in the ATD of NR2A that strongly influences the gating and zinc- dependent desensitization;(4) To test the hypothesis that transmembrane domains and their linkers to the ligand-binding domain play a critical role in the pH-sensitive gating and the allosteric interaction between the ATD and the S1/S2 domain. Relevance: This study will determine how zinc modulates a molecule called NMDA receptors. Glutamate is a molecule leaked from dying neurons that casuses more neurons to die and NMDA receptors play a critical role in this vicious circle of cell death. Zinc can reduce the activity of NMDA receptors by causing a process called "desensitization". Knowledge about "zinc-dependent desensitization" of NMDA receptors may lead to new drugs that can be used to reduce or block the cell death in stroke, seizures.